Comparative evaluation of nucleic acid-based assays for detection of Japanese encephalitis virus in swine blood samples

Comparative evaluation of nucleic acid-based assays for detection of Japanese encephalitis virus... Japanese encephalitis is an emerging mosquito-borne flaviviral zoonotic disease. The present study was undertaken with the objective of developing rapid and sensitive nucleic-acid-based assays for detection of Japanese encephalitis virus (JEV) in swine blood samples. Three nucleic-acid-based assays, viz ., reverse transcription polymerase chain reaction (RT-PCR), reverse transcription loop-mediated isothermal amplification (RT-LAMP), and real-time RT-PCR, were developed and compared in terms of their diagnostic efficacy. All three assays were found to be 100 per cent specific. The minimum detection limit of RT-LAMP and real-time RT-PCR was 12 copies/µl, while RT-PCR could detect 1.2 × 10 5 copies/µl. On comparison, RT-LAMP and real-time RT-PCR were 4-log more sensitive than RT-PCR. The applicability of the assays was evaluated by screening 135 field swine blood samples, of which 24 (17.77 %) were positive by RT-LAMP and real-time RT-PCR and only six (4.44 %) were positive by RT-PCR. The viral load in swine blood samples ranged between 2 × 10 6 and 4.8 × 10 9 copies per ml of blood by real-time RT-PCR. The comparative diagnostic sensitivity and specificity of RT-LAMP vis-à-vis real-time RT-PCR was found to be 100 %, while the sensitivity and specificity of RT-PCR vis-à-vis real-time RT-PCR was found to be 25 % and 100 %, respectively. Thus, the use of RT-PCR may cause the incidence of JEV in the swine population to be underestimated, while the real-time RT-PCR reported here is the test of choice for reference laboratories, and the newly developed one-step RT-LAMP assay will be suitable for field-level testing. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Archives of Virology Springer Journals

Comparative evaluation of nucleic acid-based assays for detection of Japanese encephalitis virus in swine blood samples

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Publisher
Springer Journals
Copyright
Copyright © 2015 by Springer-Verlag Wien
Subject
Biomedicine; Virology; Medical Microbiology; Infectious Diseases
ISSN
0304-8608
eISSN
1432-8798
D.O.I.
10.1007/s00705-015-2385-3
Publisher site
See Article on Publisher Site

Abstract

Japanese encephalitis is an emerging mosquito-borne flaviviral zoonotic disease. The present study was undertaken with the objective of developing rapid and sensitive nucleic-acid-based assays for detection of Japanese encephalitis virus (JEV) in swine blood samples. Three nucleic-acid-based assays, viz ., reverse transcription polymerase chain reaction (RT-PCR), reverse transcription loop-mediated isothermal amplification (RT-LAMP), and real-time RT-PCR, were developed and compared in terms of their diagnostic efficacy. All three assays were found to be 100 per cent specific. The minimum detection limit of RT-LAMP and real-time RT-PCR was 12 copies/µl, while RT-PCR could detect 1.2 × 10 5 copies/µl. On comparison, RT-LAMP and real-time RT-PCR were 4-log more sensitive than RT-PCR. The applicability of the assays was evaluated by screening 135 field swine blood samples, of which 24 (17.77 %) were positive by RT-LAMP and real-time RT-PCR and only six (4.44 %) were positive by RT-PCR. The viral load in swine blood samples ranged between 2 × 10 6 and 4.8 × 10 9 copies per ml of blood by real-time RT-PCR. The comparative diagnostic sensitivity and specificity of RT-LAMP vis-à-vis real-time RT-PCR was found to be 100 %, while the sensitivity and specificity of RT-PCR vis-à-vis real-time RT-PCR was found to be 25 % and 100 %, respectively. Thus, the use of RT-PCR may cause the incidence of JEV in the swine population to be underestimated, while the real-time RT-PCR reported here is the test of choice for reference laboratories, and the newly developed one-step RT-LAMP assay will be suitable for field-level testing.

Journal

Archives of VirologySpringer Journals

Published: May 1, 2015

References

  • Viruses in boar semen: detection and clinical as well as epidemiological consequences regarding disease transmission by artificial insemination
    Guerin, B; Pozzi, N
  • Development and evaluation of SYBR Green I-based one-step real-time RT-PCR assay for detection and quantitation of Japanese encephalitis virus
    Santhosh, SR; Parida, MM; Dash, PK; Pateriya, A; Pattnaik, B; Pradhan, HK; Tripathi, NK; Ambuj, S; Gupta, N; Saxena, P; Rao, PVL

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